Centre for Infectious Diseases and Microbiology - Public Health, Westmead Hospital, Westmead, NSW, Australia.
Westmead Clinical School, The University of Sydney, Westmead, NSW, Australia.
Microb Genom. 2020 Mar;6(3). doi: 10.1099/mgen.0.000332.
, the aetiological agent of whooping cough, is re-emerging globally despite widespread vaccination. is highly infectious and, prior to vaccination programmes, was the leading cause of infant mortality. The WHO estimated that over 600 000 deaths are prevented annually by pertussis vaccination, but infection was still responsible for over 63 000 deaths globally in 2013. The re-emergence of has been linked to strains with inactive or absent major virulence factors included in vaccines such as pertactin, pertussis toxin and filamentous haemagglutinin. Thus, the molecular surveillance of currently circulating strains is critical in understanding and controlling . Such information provides data on strains to inform control measures and the identification of future vaccine antigens. Current surveillance and typing methods for rely on the availability of clinical isolates. However, since the 1990s, the majority of pertussis cases have been diagnosed by PCR, where an isolate is not needed. The rapid decline in the availability of isolates impacts our ability to monitor this infection. The growing uptake of next-generation sequencing (NGS) has offered the opportunity for culture-independent genome sequencing and typing of this fastidious pathogen. Therefore, the objective of the study was to optimize respiratory sample preparation, independent of culture, in order to type using NGS. The study compared commercial depletion kits and specimen-processing methods using selective lysis detergents. The goal was to deplete human DNA, the major obstacle for sequencing a pathogen directly from a clinical sample. Samples spiked with a clinically relevant amount of were used to provide comparison between the different methods. Commercial depletion kits including the MolYsis, Qiagen Microbiome and NEBNext Kits were tested. Previously published methods, for Saponin and TritonX-100, were also trialled as a depletion. The ratio of to human DNA was determined by real-time PCR for ERV3 and (as markers of human and DNA, respectively), then samples were sequenced using the Illumina NextSeq 500 platform. The number of human and sequenced reads were then compared between treatments. The results showed that commercial kits reduced the human DNA present, but also reduced the concentration of target . However, selective lysis with Saponin treatment resulted in almost undetectable levels of human DNA, with minimal loss of target DNA. Sequencing read depth improved five-fold in reads to . Our investigation delivered a potential protocol that will enable the public health laboratory surveillance of in the era of culture-independent testing.
百日咳是百日咳的病原体，尽管广泛接种疫苗，但仍在全球重新出现。它具有高度传染性，在疫苗接种计划之前，它是婴儿死亡的主要原因。世界卫生组织估计，每年有超过 60 万人因百日咳疫苗接种而免于死亡，但 2013 年全球仍有超过 63000 人死于感染。百日咳的重新出现与疫苗中包含的无活性或缺失的主要毒力因子（如 pertactin、百日咳毒素和丝状血球凝集素）的菌株有关。因此，目前流行菌株的分子监测对于理解和控制 至关重要。此类信息提供了有关菌株的信息，以告知控制措施和确定未来的疫苗抗原。目前对 的监测和分型方法依赖于临床分离株的可用性。然而，自 20 世纪 90 年代以来，大多数百日咳病例都是通过 PCR 诊断的，而不需要分离株。可用于监测这种感染的分离株的迅速减少影响了我们监测这种感染的能力。下一代测序（NGS）的广泛采用为这种难以培养的病原体提供了独立于培养的基因组测序和分型的机会。因此，本研究的目的是优化呼吸样本的制备，使其独立于培养，以便使用 NGS 对 进行分型。该研究比较了商业性耗竭试剂盒和使用选择性裂解去污剂的标本处理方法。其目的是耗尽人 DNA，这是直接从临床样本中测序病原体的主要障碍。用临床相关量的 接种的样本用于比较不同方法之间的差异。测试了包括 MolYsis、Qiagen Microbiome 和 NEBNext 试剂盒在内的商业性耗竭试剂盒。还尝试了以前发表的 Saponin 和 TritonX-100 方法作为耗竭剂。通过实时 PCR 测定 ERV3 和 （分别作为人 DNA 和 DNA 的标志物）的 与人类 DNA 的比值，然后使用 Illumina NextSeq 500 平台对样品进行测序。然后比较处理之间的人类和 测序读长。结果表明，商业试剂盒减少了人 DNA 的存在，但也减少了目标 的浓度。然而，Saponin 处理的选择性裂解导致人 DNA 几乎无法检测到，而目标 DNA 的损失最小。测序读长深度提高了五倍，达到 。我们的研究提供了一种潜在的方案，将使公共卫生实验室能够在独立于培养的检测时代对 进行监测。
